Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II).
The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1mol of histidine was modified per mol of CPT-II. When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity. The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely. It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated COT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced. Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine.
Nic a'Bháird
N
N
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143 USA.
Yankovskaya
V
V
Ramsay
R R
RR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002333:Carnitine Acyltransferases / Q000737:chemistry* / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D004139:Dinitrofluorobenzene / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats
D004139:Dinitrofluorobenzene; D002333:Carnitine Acyltransferases; C030162:carnitine octanoyltransferase; D002334:Carnitine O-Palmitoyltransferase; D004047:Diethyl Pyrocarbonate